Download IN VITRO AGAINST MCF-7 AND HeLa CELL LINE CULTURES

Academic Sciences
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 4, Issue 1, 2012
Research Article
IN VITRO CYTOTOXIC ACTIVITIES OF CALOTROPIS PROCERA LATEX AND FLOWER EXTRACTS
AGAINST MCF-7 AND HeLa CELL LINE CULTURES
PUSAPATI MADAN RANJIT*, KRISHNA PRIYA M, SILPA P, NAGALAKSHMI V, ANJALI M, GIRISH K, AND
Y.A.CHOWDARY
Department of Pharmaceutical Biotechnology, NRI College of Pharmacy, Pothavarappadu Village, Agiripalli Mandal, Krishna District, A.P,
India. Email: [email protected]
Received: 12 Feb 2011, Revised and Accepted: 18 May 2011
ABSTRACT
Calotropis procera is a wild growing plant with multifarious medicinal properties. The present study was carried out to evaluate the effect of dried
latex (DL) and flowers of C.procera and its ethanolic extracts against MCF-7 and HeLa cell line cultures. The MTT assay developed by Mosmann, it
was modified and used to determine the inhibitory effects of test compounds on cell growth in vitro. Different concentrations of test compounds
were used (Sample: 1 DL extract, Sample: 2 Flower extract) (4, 8, 16, 32, 64, 128, 256 and 512 µg/ml) in triplicates to achieve a final volume of 100
µl and then cultured for 48 hr. The standard (Tamoxifen) was prepared as 1.0 mg/ml concentration stock solutions in PBS. Culture medium and
solvent were used as controls. The standard drug tamoxifen inhibits 60.46% breast cancer (MCF-7) cells when treated at 512 μg/ml concentration.
At initial concentrations of the standard drug, the percent inhibition was reduced whereas both the samples: 1 and 2 displayed a dose-dependent
inhibition at the tested concentrations. The ethanolic extract of DL and flowers showed cytotoxic properties against both MCF-7 and HeLa cells in a
dose dependent manner.
Keywords: Calotropis procera, MCF-7 cell line, HeLa cell line, In vitro cytotoxicity
INTRODUCTION
In Vitro Cytotoxicity Studies
Calotropis procera (Ait.)R.Br. (Asclepiadaceae) commonly known, as
Arka’ is a popular medicinal plant found throughout the tropics of
Asia and Africa and is used in many traditional systems of medicine.
Important factors of the various parts of this plant have been widely
reported. In traditional medicinal system, it has been used for a
variety of diseases condition that include leprosy, ulcers, tumors and
piles1.The plant has also been used as an emetic and purgative.
Besides, it has been shown to exhibit spasmogenic and carminative
properties1, 2. The flowers and every part of this plant contain latex
in abundance3. Different parts of the Calotropis procera are used in
treatment of various diseases in folk medicine, and their effects were
confirmed by various scientific experiments4. Besides, the
chloroform extract of the roots has been shown to possess antiinflammatory and analgesic properties5. The anti-inflammatory and
analgesic properties were exhibited by the latex of the plant which is
abundant in the aerial parts6, 7. The latex also exhibits free radical
scavenging and antioxidant properties8, 9, in addition to its
application as an antidote for snake poisoning10. It also showed
antitumor activity tested in trials using different tumor cell lines and
it was able to inhibit the development of cell lines HL-60 (leukemia),
HCT-8 (colon cancer), MDA-MB-435 (Breast cancer) and F295 (cell
cancer of the brain) when analyzed11. This study showed the in vitro
cytotoxic activities of ethanolic extract of dry latex and flowers of
Calotropis procera on MCF-7 and HeLa cell lines.
Maintenance of Cell Lines
MATERIALS AND METHODS
Plant Material
Breast cancer (MCF-7) cells and carcinoma of cervix (HeLa) cells were
maintained in Dulbecco’s modified essential medium (DMEM)
supplemented with 4.5 g/L glucose and 2 mM L-glutamine and 5% foetal
bovine serum (FBS, Growth medium) at 37°C in 5% CO2 incubator.
Cell culture
Carcinoma of cervix (HeLa), breast, melanoma and thyroid cells
were maintained in Dulbecco’s modified essential medium (DMEM)
supplemented with 4.5 g/L glucose, 2 mM L-glutamine and 5% fetal
bovine serum (FBS, Growth medium) at 37°C in 5% CO2 incubator.
MTT assay
The MTT assay developed by Mosmann was modified and used to
determine the inhibitory effects of test compounds on cell growth in
vitro 12. In brief, the trypsinized cells from T-25 flask were seeded in
each well of 96-well flat-bottomed tissue culture plate at a density of
5x103 cells well in growth medium and cultured at 37°C in 5% CO2 to
adhere. After 48hr incubation, the supernatant was discarded and
the cells pretreated with growth medium, subsequently mixed with
different concentrations of test compounds (Sample: 1 DL extract,
Sample: 2 Flower extract) (4, 8, 16, 32, 64, 128, 256 and 512 µg/ml)
in triplicates to achieve a final volume of 100 µl and then cultured
for 48 hr. The standard (Tamoxifen) was prepared as 1.0 mg/ml
concentration stock solutions in PBS. Culture medium and solvent
were used as controls.
The flowers and latex were collected in month of September from-2010
out fields of Agiripalli Mandal, Krishna district, identified and herbarium
specimen was deposited in the department of Pharmacognosy with
specimen
No.NRI/COL/P.COG/1/PF
(Flowers)
and
NRI/COL/P.COG/2/PL (Latex). The latex was collected from the aerial
parts of the plant growing in wild, it was air dried under shade at
ambient temperature and ground to small granules of dried latex (DL).
Each well then received 5 µl of fresh MTT (0.5mg/ml in PBS)
followed by incubation for 2hr at 37°C. The supernatant growth
medium was removed from the wells and replaced with 100 µl of
DMSO to solubilize the colored formazan product. After 30 min
incubation, the absorbance (OD) of the culture plate was read at a
wavelength of 570 nm on an ELISA reader, Anthos 2020
spectrophotometer.
The shade-dried powder of flowers was subjected to extraction in
soxhlet extractor with 70% EtOH for 70 hours (extract yield: 9%)
and Dry latex (DL) was macerated with 70% EtOH and extract
(extract yield: 4.5%) was collected. Both extracts were evaporated
to dryness and stored at 4 ◦ C until used.
Carcinoma of breast
Preparation of extracts
RESULTS AND DISCUSSION
Results in table 1 showed that the standard drug tamoxifen inhibits
60.46% breast cancer (MCF-7) cells when treated at 512 μg/ml
concentration. At initial concentrations of the standard drug, the
Ranjit et al.
percent inhibition was reduced whereas both the samples: 1 and 2
displayed a dose-dependent inhibition (Table 2 and 3) at the tested
concentrations. However, more than 50% inhibition was not
observed in both the cases.
Carcinoma of cervix
It has been observed that the percentage inhibitory activity of the
two samples 1 and 2 tested against carcinoma of cervix (HeLa cells)
yielded much lower percentage inhibition than the standard drug
tamoxifen. Results in table 4 showed that more than 50% inhibition
was observed at 128 μg/ml concentration of the standard drug,
whereas both the samples at highest tested dose displayed 41-43%
inhibition only (Table 6 and 5).
The present study was carried out to evaluate the in vitro cytotoxic
activities of ethanolic extract of both DL and flower of C.procera on
MCF-7 and HeLa cells. However, from the graphs (Fig. 1, Fig. 2) it
was evident that a dose-dependent relationship was observed for all
the samples under study. This shows that the samples have some
growth inhibitory characteristics against carcinoma of breast cancer
and cervix cell line (Fig. 3). Inhibitory data as well as comparative
graphs are also provided. Smit et al reported that C. procera flower
extract showed the potent in vitro cytotoxic activity, which is
comparable to standard anticancer drug cisplatin13. Another report
explained that latex of C. procera exhibits cytotoxic properties like
standard
anticancer
drugs
podophyllotoxin
and
cyclophosphamide14. The latex has also showed cytotoxicity
corroborated by morphological alterations in leukemia cells, such as
chromatin condensation, DNA fragmentation and cell volume
Int J Pharm Pharm Sci, Vol 4, Issue 1, 66-70
reduction15, 16. Another study reported that, X15-myc transgenic
mice treated with DL (400mg/kg) for a period of 15 week, protected
mice from malignant changes occurring in liver while sinusoidal
architecture and cellular integrity were slightly disrupted as
compared to normal and hydropic changes were observed. DL
produced a significant decrease in the serum vascular endothelial
growth factor (VEGF) levels of X15-myc transgenic mice. The
methanolic extract (ME) of DL was induced cell death in cell lines
viz., Huh-7 and COS-1 cells. It was evaluated by using tetrazolium
(MTT) assay. The ME was subjected to silica gel G step column
chromatography using a combination of non-polar and polar
solvents and 11 fractions were obtained. Out of 11 fractions, fraction
8 exhibited potent cytotoxic effect on both the cell lines. However, a
marginal effect on the killing of non-cancerous cell lines suggested a
high degree of selectivity for transformed cells. Such differential
killing of cancerous cells could relate to their altered metabolic
status and/or membrane properties. The cytotoxic effect of DL was
accompanied by intracellular fragmentation of target cell DNA17.
Our study reported that ethanolic extract of DL and flowers showed
cytotoxic properties against both MCF-7 and HeLa cells in a dose
dependent manner.
As per our knowledge, our studies reported that both ethanolic
extracts of DL and flower showed cytotoxic effects on estrogen
positive cell line (i.e.MCF-7) and carcinoma of cervix (i.e. HeLa cells).
The cytotoxic activities of C. procera may be due to the presence of
various phytoconstituents such as cardenolides, flavonoids and other
constituents18-21. However, the molecular mechanism for such an
effect needs further evaluation.
Table 1: Percentage of inhibition of breast cancer (MCF-7) cells with respect to standard concentration of tamoxifen
Conc(μg/ml)
Observed OD
4
1.445
8
1.361
16
1.149
32
1.106
64
1.093
128
0.976
256
0.627
512
0.588
Control OD: 1.487
% Viability = Observed OD/Control OD * 100
% Inhibition = 100%viability
%Viability
97.18
91.53
77.27
74.38
73.50
65.64
42.17
39.54
%Inhibition
2.82
8.47
22.73
25.62
26.50
34.36
57.83
60.46
Table 2: Percentage of inhibition of breast cancer (MCF-7) cells with respect to test sample: 1(DL extract)
Conc. (μg/ml)
Observed OD
4
1.361
8
1.287
16
1.202
32
1.165
64
1.035
128
0.987
256
0.942
512
0.802
Control OD: 1.487
% Viability = Observed OD/Control OD * 100
% Inhibition = 100%viability
%Viability
91.53
86.55
80.83
78.35
69.60
66.38
63.35
53.93
Conc. (μg/ml)
Observed OD
4
1.305
8
1.211
16
1.184
32
1.133
64
0.933
128
0.961
256
0.912
512
0.838
Control OD: 1.487
% Viability = Observed OD/Control OD * 100
% Inhibition = 100% viability
%Viability
87.76
81.44
79.62
76.19
66.78
64.63
61.33
56.36
%Inhibition
8.47
13.45
19.17
21.65
30.40
33.62
35.65
46.07
Table 3: Percentage of inhibition of breast cancer (MCF-7) cells with respect to test sample: 2 (Flower extract)
%Inhibition
12.24
18.56
20.38
23.81
33.22
35.37
38.67
43.64
67
Ranjit et al.
Int J Pharm Pharm Sci, Vol 4, Issue 1, 66-70
Table 4: Percentage of inhibition of carcinoma of cervix (HeLa cells) with respect to standard concentration of tamoxifen
Conc(μg/ml)
Observed OD
4
2.097
8
2.084
16
1.988
32
1.564
64
1.249
128
1.015
256
0.954
512
0.815
Control OD: 2.254
% Viability = Observed OD/Control OD * 100
% Inhibition = 100% viability
%Viability
93.03
92.46
88.20
69.39
55.41
45.03
42.32
36.16
Conc(μg/ml)
Observed OD
4
2.152
8
1.956
16
1.889
32
1.755
64
1.602
128
1.445
256
1.369
512
1.265
Control OD: 2.254
% Viability = Observed OD/Control OD * 100
% Inhibition = 100% viability
%Viability
95.47
86.78
83.81
77.86
71.07
64.11
60.74
56.12
Conc(μg/ml)
Observed OD
4
2.164
8
1.789
16
1.651
32
1.502
64
1.466
128
1.401
256
1.388
512
1.311
Control OD: 2.254
% Viability = Observed OD/Control OD * 100
% Inhibition = 100% viability
%Viability
96.01
79.37
73.25
66.64
65.07
62.16
61.58
58.16
%Inhibition
6.97
7.54
11.08
30.61
44.59
54.97
57.68
63.84
Table 5: Percentage of inhibition of carcinoma of cervix (HeLa cells) with respect to test sample: 1(DL extract)
%Inhibition
4.53
13.22
16.19
22.14
28.93
35.89
39.26
43.88
Table 6: Percentage of inhibition of carcinoma of cervix (HeLa cells) with respect to test sample: 2 (Flower extract)
%Inhibition
3.99
20.63
26.75
33.36
34.96
37.84
38.42
41.84
Fig. 1: Shows inhibition of breast cancer cells (MCF-7) by sample-1 and 2 with comparison to standard tamoxifen
Fig. 2: Shows inhibition of carcinoma of cervix (HeLa cells) by sample-1 and 2 with comparison to standard tamoxifen
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Ranjit et al.
Int J Pharm Pharm Sci, Vol 4, Issue 1, 66-70
Fig. 3: Shows A) MCF-7 cells before treatment showing actively dividing cells, B) MCF-7 cells after treatment with sample-1 at 512μg/ml
concentration incubated for 24hrs showing uncharacterized bodies, C) HeLa cells before treatment with samples or drug showing active
cells grown in DMEM in a 96 well plate, D) HeLa cells after treatment with sample-1 at highest tested dose (512 μg/ml) showing
unchracterized bodies. (All photographed viewed under inverted microscope)
ACKNOWLEDGEMENT
The authors are thankful to the management and principal of NRI
College of Pharmacy, Pothavarappadu, Agiripalli mandal, Krishna
Dist, A.P, India, for providing the research facilities and also to the
Research Gateway for Biosciences, Dwarakanagar, Visakhapatnam,
A.P. India, for carryingout the cell line culture work.
9.
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